Ion channel targeted drug discovery has been hampered by the unavailability of cell-based assays that utilize physiological and pharmacological relevant measurements to characterize compound activity. This project will develop an assay to measure intracellular chloride ion and pH as direct physiological measurements to interrogate the chloride ion channels and proton transporter(s) for GI drug discovery. The Phase I of this proposal will focus on the synthesis, characterization and validation of the chloride ion sensing indicator using T84 cells. The Phase II of this proposal will focus on the development of the pH sensing indicator, integration of the pH sensing assay with the Cl assay;validation of the joint chloride and pH assay using T84 cells by delineating the intracellular coupling dynamics of [Cl-]i and pHi for GI drug discovery;and scaling up the manufacturing of the assays for commercialization. PUBLIC HEALTH RELEVANCE: Ion channels of chloride and proton transports are the two predominant drug discovery gastrointestinal (GI) target sites for the treatments of diarrhea, constipation and gastric reflux. Cytoptics Corporation proposes to develop a new generation of chloride ion and pH sensing indicators to measure intracellular chloride ion concentrations, [Cl-]i, and pHi simultaneously for cell-based GI drug discovery. This Phase I proposal will focus on the 1) synthesis and spectral characterization of chloride ion sensing indicator;2) development of the loading protocols of the indicator;and 3) physio-pharmacological measurements of [Cl-]i in T84 cells. This will enable a chloride ion sensing assay kit to be readily marketed for GI drug discovery and development following the completion of the Phase I. The Phase II of this proposal will focus on the development of the pH sensing indicator, integration of the pH sensing assay with the Cl assay;validation of the joint chloride and pH assay using T84 cells by delineating the intracellular coupling dynamics of [Cl-]i and pHi for GI drug discovery;and scaling up the manufacturing of the assays for commercialization.